This is a request for an extension of a Merit Award. Work during this grant period has shown that there are three major mechanisms controlling ENaC activity. The first is control of ENaC transcription, translation, and trafficking; the second is control of the activity of channels at the membrane; and the third is regulation of ENaC retrieval, recycling, and degradation. Although these appear to be disparate control mechanisms, the underlying mechanism that all the pathways appear to have in common as a final step, is that they depend upon membrane levels of inositol lipid phosphate modulators of ENaC function. Aldosterone, activation of G-protpin-linked receptors, and growth factors, among others, all alter ENaC activity by increasing or decreasing phosphatidylinositolphosphates. The specific aims for the proposed extension period will investigate the three major mechanisms that regulate Na transport. The hypothesis is that inositol lipids play a role in all aspects of ENaC regulation: PIPS plays a role in ENaC trafficking to the membrane; PIP2 and PIPS binding at the membrane affects ENaC gating; and the extent of PIP2 and PIPS binding determines access to ubiquitin ligase that promotes retrieval and degradation of ENaC. The aims are:(1) continue to examine the regulation of Na channels in the membrane by inositol lipids and inositol lipid kinases and phosphatases; (2) determine the binding sites for inositol lipids on ENaC; (3) investigate whether changes in the level of PIP2 and PIPS in the membrane affects ENaC ubiquitinylation and the fate of ENaC retrieval from the membrane; (4) examine whether RhoA-mediated insertion of ENaC into the surface membrane depends upon inositol lipids; and (5) determine if PIPS is necessary for ENaC trafficking from the ER and Golgi. This award has lead to 32 peer-reviewed articles, 7 review articles, 3 book chapters, and 1 book. These studies together suggest that one primary mechanism for regulating ENaC is by alteration of anjonic phospholipids and that the receptor-mediated and hormonal regulation of ENaC works through a variety of signaling pathways, but many of these pathways finally alter ENaC activity by regulating the formation or degradation of anionic phospholipids. Thus, an understanding of how membrane levels of PIP2 and PIPS are regulated will be important to understand how ENaC is regulated and, consequently, how total body sodium and blood pressure is regulated.